ISSN 0722-4060, Volume 33, Number 6

ISSN 0722-4060, Volume 33, Number 6 This article was published in the above mentioned Springer issue. The material, including all portions thereof, is protected by copyright; all rights are held exclusively by Springer Science + Business Media. The material is for personal use only; commercial use is not permitted. Unauthorized reproduction, transfer and/or use may be a violation of criminal as well as civil law. Polar Biol (2010) 33:869–875 Author's personal copy DOI 10.1007/s00300-009-0758-3 SHORT NOTE Phylogenetic diversity of culturable bacteria from Antarctic sandy intertidal sediments Yong Yu · Huirong Li · Yinxin Zeng · Bo Chen Received: 14 September 2009 / Revised: 8 December 2009 / Accepted: 9 December 2009 / Published online: 24 December 2009 © Springer-Verlag 2009 Abstract The diversity of culturable bacteria associated and frozen algae at Adelie Land near the South Pole. Then, a with sandy intertidal sediments from the coastal regions of wide variety of strains have been isolated from various the Chinese Antarctic Zhongshan Station on the Larsemann marine or terrestrial environments of Antarctica (Friedmann Hills (Princess Elizabeth Land, East Antarctica) was inves- 1993; Bowman et al. 1997a; Nichols et al. 1999; Brambilla tigated. A total of 65 aerobic heterotrophic bacterial strains et al. 2001; Mergaert et al. 2001; Van Trappen et al. 2002; were isolated at 4°C. Microscopy and 16S rRNA gene Helmke and Weyland 2004; Michaud et al. 2004; Laybourn- sequence analysis indicated that the isolates were domi- Parry and Pearce 2007; Babalola et al. 2009). In many cases, nated by Gram-negative bacteria, while only 16 Gram-posi- the strains isolated were proven to be unknown taxa (Sch- tive strains were isolated. The bacterial isolates fell in Wve mann et al. 1997; Bowman et al. 1997b; Bowman et al. phylogenetic groups: Alpha- and Gammaproteobacteria, 1998; Labrenz et al. 2000; McCammon and Bowman 2000; Bacteroidetes, Actinobacteria and Firmicutes. Based on Bowman and Nichols 2002; Sheridan et al. 2003; Yakimov phylogenetic trees, all the 65 isolates were sorted into 29 et al. 2003; Van Trappen et al. 2004; Bowman and Nichols main clusters, corresponding to at least 29 diVerent genera. 2005; Yi et al. 2005; Yi and Chun 2006; Lee et al. 2007; Yu Based on sequence analysis (<98% sequence similarity), et al. 2008; Labrenz et al. 2009). In contrast to marine or ter- the Antarctic isolates belonged to at least 37 diVerent bacte- restrial environments, the marine–land interface (intertidal) rial species, and 14 of the 37 bacterial species (37.8%) rep- environment around Antarctica seems to be ignored by resented potentially novel taxa. These results indicated a microbiologists. Peck et al. (2006) recently reviewed the high culturable diversity within the bacterial community of environmental constraints on life history strategies in Ant- the Antarctic sandy intertidal sediments. arctic marine, intertidal and terrestrial environments. They conclude that the intense seasonal scouring by ice, winter ice Keywords Heterotrophic bacteria · Antarctica · encasement, high UV radiation, heavy salinity and tempera- Intertidal sediments · Phylogenetic analyses ture Xuctuations make Antarctic intertidal zone, possibly the world’s most physically disturbed environment. The extreme conditions do prevent resident macro-algal develop- Introduction ment, but do not prevent diatoms, bacterial and algal Wlms thriving (Peck et al. 2006). A wide range of infauna was also Prokaryotes dominate many Antarctic ecosystems and con- found to inhabit Antarctic intertidal zone (Waller 2008). trol most of the biological Xux of carbon, nutrients and However, information on the biodiversity and community energy. McLean (1918) Wrst isolated bacteria from snow, ice structure of bacteria in this area is still lacking. Our study presents the phylogenetic analysis of 65 bacterial strains isolated from sandy intertidal sediments collected oV the Y. Yu (&) · H. Li · Y. Zeng · B. Chen coastal regions of the Chinese Antarctic Zhongshan Station SOA Key Laboratory for Polar Science, Polar Research Institute of China, on the Larsemann Hills (Princess Elizabeth Land, East Ant- 200136 Shanghai, People’s Republic of China arctica) in order to obtain a preliminary understanding of e-mail: [email protected] bacterial community composition in this environment. 123 870 Author's personal copy Polar Biol (2010) 33:869–875 Materials and methods of denaturation at 95°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 2 min, with a Wnal exten- Sediment samples sion at 72°C for 10 min. The PCR products were pooled and puriWed with the gel extraction kit (Watson, Shanghai, A total of four sandy intertidal sediment samples were col- China), and ligated into the pMD 18-T vectors (TaKaRa, lected from the coastal regions of the Chinese Antarctic Japan). The hybrid vectors were used for transformation Zhongshan Station on the Larsemann Hills (Princess Eliza- into Escherichia coli DH5 competent cells. Recombinants beth Land, East Antarctica) during the 23rd Chinese were selected using Luria–Bertani (LB) indicator plates National Antarctic Research Expedition in March 2007. containing 100 g of ampicillin per ml, 80 g of X-Gal These surWcial sediments (0–3 cm) were sampled by small (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) per ml, sterilized shovels and stored in sterilized plastic bags 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). (250 ml). Samples were transported to the laboratory at White clones were sequenced by ABI PRISM 3730 temperatures between 0 and 4°C. sequencer at Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Isolation of bacterial strains Phylogenetic analysis In laboratory, 1 g of wet sediment sample was mixed with 99 mL of sterilized seawater supplemented with 10 glass The sequences, 1,427–1,515 nt, depending on the isolate, beads (diameter 2–3 mm) and shaken at 4°C for 1 h at 300 were compared with the data available in the RDPII (Ribo- repetitions/min. The culture was further diluted (1:10) with somal Database Project II) using the sequence match tool, sterilized seawater and spread onto three diVerent marine to determine the relative phylogenetic positions. The identi- agars. These included 1/10-strength marine R2A agar Wcation of phylogenetic neighbors and the calculation of (Suzuki et al. 1997), 1/10-strength marine 2216 agar pairwise 16S rDNA sequence similarities were achieved (Difco) and natural seawater agar. The plates were incu- using the EzTaxon server (Chun et al. 2007). In addition, bated in the dark at 4°C for up to 8 weeks. Colonies from the sequences were compared to the sequences from within various agar plates were picked on the basis of diVering the NCBI database (http://www.ncbi.nlm.nih.gov/) using colony morphologies. Isolates were obtained in pure culture BLASTN. Sequences were aligned using Clustal X1.8 after three successive transfers to fresh agar medium and (Thompson et al. 1997), with most closely homologous stored at ¡80°C in marine 2216 broth (Difco) supple- bacterial type strains 16S rDNA sequences retrieved from mented with 30% (v/v) glycerol. The Gram reaction was GenBank. Alignments were edited manually using BioEdit examined with a phase-contrast microscope (Eclipse 80i; Sequence Alignment Editor version 5.0.9 (Hall 1999) and Nikon) following the standard Gram procedure (Murray regions of ambiguous alignment were removed. The phylo- et al. 1994). genetic tree was constructed using the neighbor-joining method (Saitou and Nei 1987) with Kimura 2-state parame- 16S rDNA ampliWcation and sequencing ter and pairwise-deletion model analyses implemented in the program MEGA version 4 (Tamura et al. 2007). The Total genomic DNA for 16S rDNA ampliWcation was iso- resultant tree topologies were evaluated by bootstrap analy- lated from 1 mL of bacteria grown to late log phase in sis based on 1,000 replicates. DNA sequences were depos- marine 2216 broth (Difco) and puriWed by kit according to ited to GenBank under Accession numbers FJ195991, the manufacturer’s instruction (BioDev, Beijing, China). FJ195992, FJ195993, FJ195994, FJ195995, FJ195996, Nearly full-length 16S rRNA gene was ampliWed by PCR FJ195997, FJ195998, FJ195999, FJ196000, FJ196001, using universal primers 8f (5Ј-AGA GTT TGA TCC FJ196002, FJ196003, FJ196004, FJ196005, FJ196006, TGGCTC AG-3Ј) and 1492r (5Ј-GGT TAC CTT GTT FJ196007, FJ196015, FJ196016, FJ196017, FJ196018, ACG ACT T-3Ј) (Lane 1991). The PCR mixtures (Wnal vol- FJ196019, FJ196020, FJ196021, FJ196022, FJ196023, ume, 50 L) contained 100 ng of the extracted DNA as a FJ196024, FJ196027, FJ196028, FJ196029, FJ196030, template, 5.0 l of 10£ PCR buVer (Sangon, Shanghai, FJ196031, FJ196032, FJ196033, FJ196050, FJ196053, China), each deoxynucleoside triphosphate at a concentra- FJ889642, FJ889643, FJ889645, FJ889646, FJ889647, tion of 40 M, 0.2 M of each primer, and 1 U Taq DNA FJ889648, FJ889649, FJ889650, FJ889653, FJ889654, polymerase (TaKaRa, Japan). PCR ampliWcation was per- FJ889655, FJ889657, FJ889658, FJ889659, FJ889660, formed with an Eppendorf Mastercycler Gradient (Eppen- FJ889664, FJ889665, FJ889666, FJ889667, FJ889668, dorf, Germany), and the following program was used: FJ889669, FJ889670, FJ889671, FJ889672, FJ889674, initial denaturation at 95°C for 4 min followed by 25 cycles FJ889675, FJ889676, FJ889677 and GQ496083. 123 Polar Biol (2010) 33:869–875 Author's personal copy 871 99 Cluster Marinobacter psychrophilus 100 Marinobacter Cluster Marinobacter maritimus

Load more